X chromosome inactivation (XCI) is a developmental program of sex chromosome dosage compensation achieved by transcriptional inactivation of one of the two X chromosomes during female mammalian embryonic development. XCI is a dramatic example of chromatin remodeling and the widespread and tightly regulated change in gene expression due to XCI can be recapitulated ex vivo during the differentiation of embryonic stem (ES) cells. XCI represents a powerful model system for dissecting the molecular relationship between pluripotency and chromatin state, but the mechanisms involved are still largely unknown. The studies described here explore coupling of developmental state to XCI status and another great unknown of XCI, the key mechanisms that contribute to silencing of the X chromosome. Aim 1. Determine if deletion of a putative Xist silencer has effects on XCI. Published data suggests that binding of ES cell transcription factors to the first intron of the Xit gene may be a regulatory mechanism for suppressing X inactivation in ES cells or reversing XCI in somatic cells. In the absence of an ectopic X-inactivation phenotype due to deletion, this region is being tested for roles in X reactivation, interaction with another XCI-related gene, and control of chromatin composition via pluripotency transcription factor binding}. Aim 2. Identify factors that are required for transcriptional silencing of the X chromosome. This aim will involve a high-throughput screen to identify genes that are required to maintain the silent state of the X. Since these mechanisms are known to be dysregulated in female progeria patient fibroblast cells (Shumaker et al., 2006) this disease cell model will be used to characterize gene hits for their role in maintenance of healthy genome-wide chromatin structure. In women dysregulation of XCI is observed in normal aging and it has been suggested to have a direct role in accelerated aging;in telomerase-deficient mouse model, telomere shortening is paralleled by dysregulation of X inactivation (Shoeftner et al., 2009). Most strikingly, changes in inactive X chromatin in cells of female Hutchinson-Gilford Progeria Syndrome patients precede hallmark pathological changes in nuclear shape (Shumaker et al., 2006). For this strong evidence linking X inactivation to aging, we believe that these studies designed to identify molecular factors involved in X inactivation are a novel approach to aging research that will open avenues in the highly sought-after areas of nuclear organization and its relationship to gene expression changes. PUBLIC HEALTH RELEVANCE: How does the mammalian developmental program generate such remarkable diversity of tissues when each cell harbors essentially the same genetic sequence material? Cell identity is dictated by gene expression, which is in turn controlled by the way genes are packaged on the chromosome-level and we are searching for regulators of this process using the X chromosome as a model. This research has potential to lead to the ability to re-package whole networks of genes and manipulate cell identity (to generate organs for transplant) or quiet vast networks of genes (to slow tumor growth), and may help us to understand how control of gene expression changes with age.